2-amino-4,6-diphenethylamino-s-triazine as active serum cholesterol lowering agent

ABSTRACT

The present invention relates to a valuable medicament for oral administration containing 2-amino-4,6-diphenethylamino-s-triazine as an effective ingredient. The drug of this invention is useful for reducing serum cholesterol level and for preventing arteriosclerosis.

United States Patent [191 Irikura et al.

11] 3,821,378 June 28, 1974 2-AMINO-4,6-DIPHENETHYLAMINO-S- TRIAZINE AS ACTIVE SERUM CHOLESTEROL LOWERING AGENT [22] Filed: Sept. 1, 1972 [2]] Appl. No: 285,680

Related US. Application Data [63] Continuation-impart of Ser. No. 134,394. April 15,

1971, abandoned.

[52] US. Cl. 424/249 [51} Int. Cl, A6lk 27/00 [58] Field of Search 424/249 [56] Q References Cited OTHER PUBLICATIONS Suter et al., Helvetica Chimica Acta, Vol. 48, 1965, pages 1940-1944.

Primary Examiner-Jerome D. Goldberg Attorney, Agent, or Firm-Toren, McGeady and Stanger [57] ABSTRACT The present invention relates to a valuable medicament for oraladministration containing 2-amino-4,6- diphenethylamino-s-triazine as an effective ingredient. The drug of this invention is useful for reducing serum cholesterol level and for preventing arteriosclerosis.

1 Claim, N0 Drawings 2-AMINO-4,6-DIPHENETHYLAMINO-S-TRIAZINE AS ACTIVE SERUM CHOLESTEROL LOWERING AGENT This application is a continuation-in-part of my copending application Ser. No. 134394 filed on April 15, 1971, now abandoned.

This invention relates to pharmaceutical compositions; more particularly it relates to pharmaceutical compositions which may be of use for the reduction of serum cholesterol and/or the suppression of arteriosclerosis.

The compound, 2-amino-4,6-diphenethylamino-striazine, is described by Hans Suter and Hans Zutter in Helvetica Chimica Acta, 48 (8), l940-l944 (1965). These authors synthesized l2 s-triazine derivatives expecting to obtain anti-diabetic agents which could be administered orally (derivatives of s-triazine were formed by ring closure of biguanides which had been already known to reduce the blood sugar level). However, as described in the report, they found no effective anti-diabetic agents.

2-Amino-4,6diphenethylamino s-triazine was one of the 12 compounds,but no details of its preparation were given, nor was the compound shown to process any pharmacological activity.

The investigations which lead to the discovery of the present invention were also. concerned with striazine derivatives, but for reasons quite different from those of Hans Suter and Hans Zutter. Recently, it was found that certain novel derivatives of s-triazine could suppress the abnormally enhanced activity of the reticuloendothelial system of animals induced by bacterial endotoxin. It was also found that there exists a relationship between this effect on hyperfunction of the reticuloendothelial system and the therapeutic effect of the derivatives on arteriosclerosis. These novel s-triazine derivatives prevent the deposition of lipids on the arterial wall without reducing the serum cholesterol level. In order that the mechanism of the anti-arteriosclerotic action and the structure activity relationship of striazine derivatives could be a further investigated.

Other s-triazine derivatives were synthesized and tested to see whether they. had any particular actions on experimental arteriosclerosis. This investigation revealed the unexpected fact that some of the s-triazine derivatives shown the ability to reduce the serum cholesterol level. In particular, it was found that 2-amino- 4,6-diphenethylamino-s-triazine suppresses the biosynthesis of cholesterol in the liver, reduces the serum cholesterol level and prevents the experimental arteriosclerosis.

This compound is the first s-triazine derivative, which has been found to possess such pharmaceutical activity.

Before the compound of this invention can be used medically, it must, of course, be formed into a pharmaceutical composition by association with a suitable pharmaceutical vehicle.

The term pharmaceutical is used herein to exclude any possibility that the nature of the vehicle, considered of course, in relation to the route by which the composition is intended to be administered, could be harmful rather than beneficial.

The choice of a suitable mode of presentation, together with an appropriate vehicle, is believed to be within the competence of those accustomed to the preparation of pharmaceutical formulations.

Accordingly this invention provides a pharmaceutical composition which comprises 2-amino-4',6- diphenethylamino-s-triazine in association with a suitable pharmaceutical vehicle.

The compositions of this invention may be administered orally and in respect of these modes, the pharmaceutical vehicle" is preferably:

the ingestible excipient of a tablet, coated tablet, sublingual tablet or pill; the ingestible container of a capsule or cachet; the ingestible pulverulent solid carrier of a powder; or the ingestible liquid medium of a syrup, solution, suspension or elixir.

In order further to illustrate this invention but without being limited thereto, the following examples are given:

EXAMPLE 1:

Preparation of 2-amino-4,6-diphenethylamino-striazine 3.3g of 2-amino-4,6-dichloro-s-triazine were suspended in ml of water and 9.7g of phenethylamine were added dropwise, with stirring, for 2 hours. After cooling the reaction mixture, the precipitate was separated andrecrystallized from isopropanol to give 6.0g of 2-aminc-4,6-diphenethylamino-s-triazine in the form of colourless needles having a melting point of l40-l42C.

Analysis: C l-l N Calculated C% 68.24 H% 5.63 N% 25.l3 Determined 6810 6.54 25.25

EXAMPLE 2:

- Tablets are prepared by mixing and granulating in accordance with known pharmaceutical techniques the following ingredients.

Ingredient: mg/tablet 2-Amino-4,6-diphenethylaminos-triazine v 50 Lactose 7 Crystalline cellulose 25 Potato starch l5 Magnesium stearate 0.5 Hydroxypropyl cellulose 2.5

EXAMPLE 3:'

Tablets are prepared by mixing and granulating in accordance with known pharmaceutical techniques, for example using the following ingredients.

Capsules are prepared in accordance with known pharmaceutical techniques from the following ingredients.

EXAMPLE Capsules are prepared in accordance with known pharmaceutical techniques, for example using the following ingredients. 0

EXAMPLE 6: g a One pulverulenta is prepared in accordance with known pharmaceutical techniques from the following ingredients.

EXAMPLES:

Granules are prepared in accordance with known pharmaceutical techniques from the following ingredients.

Ingredient:

2-Amino-4.6-diphenethylaminos'triazine 200mg Lactose 550mg Potato startch 150mg Crystalline cellulose 100mg 3% Polyvinyl alcohol 205/H,O proper quantity EXPERIMENT A The effect of 2-amino-4,6-diphenethylamino-striazine on the serum cholesterol level in mice fed a cholesterol-enriched diet.

Male dd-strain mice, 8 weeks old and weighing 20 23g, were employed. These were divided into three groups. One group (the normal group) was fed a basal diet, the second group (the control group) was fed a cholesterol-enriched diet (the basal diet with the addition of 2 percent of cholesterol and 0.1 percent of cho lic acid), whilst the third group was fed the basal diet with the addition of 2 percent of cholesterol, 0.1 percent of cholic acid and 0.0714 percent of 2-amino- 4,6-diphenethylamino-s-triazine. All the mice were fed those diets for 14 days and were decapitated on the 14th day.

Whole blood was collected and the total choresterol in the serum was determined by the Zak and Henly method; the results are shown in Table I. This Table lngredem' mg/Pulve' Ema clearly shows that 2-ammo-4,6-drphenethylammo-s- 2-Ami no-4,6-diphenethylamino triazine significantly suppresses the increase in the serum cholesterol level in the mice fed a cholesterol- Lactose 450 enrrcheddlet. v

TABLE] (3) Number Increase Food Dose of Total chol- Rate of of mice of body intake choleesterol in suppressin weight sterol serum (mg%) sion Group group (gm) (gm/kg/day) (gm/kglday) (meantS.D.)

Nonnal 9 0.6 160.6 0 173.3:225 Control l0 2.7 189.7 3.794 270.2:3L9 Z-Amino- 4,6-diphenethyl- 8 LI 163.2 3.264 2l5.4i30.3 56.5 lamino-striazine Significantly lower than the control group (P 0.005). (a) The rate of suppression was calculated by the following equation:

(Total serum cholesterol (Total serum choleof mice receiving Z-am ino- Rate of sterol of controls) 4,6-diphenethylamino-ssuppre- W X 100 ssion (Total serum chole- (Total serum cholesterol W Y 32! 9? 31 5 .QZDQFE'E E W EXAMPLE 7: EXPERIMENT B A suspension for oral administration is prepared in accordance with known pharmaceutical techniques from the following ingredients.

Ingredient:

2-Amino-4,6-diphenethylamino- Efi'ect of 2-amino-4,6-diphenethylamino-s-triazine on the serum cholesterol level in normal rats.

Male Wistar strain rats, 10 weeks old and weighting 236 300g, were employed. The tests were conducted with three groups of rats; these three groups were fed diets containing 0.0 percent (as control), 0.1 percent and 0.3 percent of 2-amino4,o-diphenethylamino-striazine respectively. The rats were all fed for 14 days and decapitated on the 14th day. Their serum cholesterol levels were then determined as in Experiment A abqveThe re ults. 9 1 9 in Tabl The results are shown in Table Hi; this Table clearly shows th at 2 -amino-4,6-diphenethylamino-s-triazine Significantly lower than control group (P 0.025)

EXPERIMENT C Suppression of the bio-synthesis of choresterol in the liver of rats by 2-amino-4,6-diphenethylamino-striazine.

Male Wistar strain rats which were fed for 7 days 2 pound. On the 8th day, these rats were given 1 ml per 200g body weight of l-"C acetate (20 pi. Curie/2O ,1. mole per 1 ml solution), by injection in the tail vein, and the rats were killed 60 minutes later. Their livers were separated and hydrolysed at 80C with 5 ml of 10 percent alcoholic potassium hydroxide solution per lgm of liver for 3 hours.

The solution obtained after the hydrolysis was evaporated, and the cholesterol was extracted from the residue with three ml of petroleum ether. The petroleum ether fraction was evaporated to dryness, and the residue was then dissolved in 10 ml of petroleum ether; 5 ml of this solution was put into a test-tube, evapo- TABLE III suppresses the bio-synthesis of cholesterol from acetate in the liver.

EXPERIMENT D Effect of 2-amino-4,o-diphenethylamino-s-triazine on experimental arteriosclerosis in rabbits.

v Three groups, each containing live rabbits, were used in this test. One group (the normal group) was fed a I basal diet.

The second group (the control group) was fed a cholesterol-enriched diet (the basal diet with the addition of 2 percent of cholesterol).

The third group (the test group) was fed the diet with the addition of 0.2 percent of 2-amino-4,6-diphenethylamino-s-triazine and 2 percent of cholesterol in the basal diet. All rabbits were fed for 80 days, then sacri- 30 ficed and examined. Atherosclerotic lesions were macthylamin'o-s-tn'azine, but the compound markedly decreased the cholesterol levels in the main artery and in the liver. The macroscopic examination also revealed Proportion of 2-amino- Number 4,6 diphenethylamino-sof Total dpm dprn/grn liver triazine in diet animals I (mean+S.E.)

0.0 8 l8.65Xl0 (2.72i0.97) l0 0.] 8 7.76Xl0 (0.80:.'t).l4) l0 0.3 8 -9.26Xl0 (0.76t0.l9) l0 TABLE IV Weight Choresterol Chlesterol of in liver Atheromata in main Group liver (mg/gm) Arch. Thrac. bd artery (mg/gm) om. (gm) (mean+S.D.) (graded 0 5) (mean+S.D.)

Normal 88:28 2.50:0.16 v 0 0 0 l.65:0.28 Control 1511M 2349x165 4.4 3.3 2.0 19.873247 Test H0120 1429:3132 2.5 L2 0.4 7.38:2.10"

' Significant decrease (P 0.0l) in comparison with the control group. Sigljljg t decrease (P 0.02) in comparison with the control grou p H W that the title compound suppresses the development of arteriosclerosis.

What is claimed is:

l. The method of treating the human body to reduce the serum cholesterol level comprising administering to a human being in need of said treatment from about 10 .to 500 mg. doses of 2-amino-4,6--diphenethylamino-striazine.

* i i it U NI TED STATES PATENT OFFICE CERTIFICATE OF QRRECTION ParehtfNopb"3 7 Dated June 1974 dfl Tgutoniu Irikura et a1 I Itji s:: Cettified that error appears in the above-identified patent and that gaid iggtpors Patent are hereby corrected as shown below:

heading of the patenfi insert:

" Kyorin Seiyaku Kabushiki Kaisha,

Tokyo, Japan--.

Si3flQ dJiId aemled this 22nd day of October 1974.

EAL) I I ;A1iteSt:

- M Y GIBSON JR. c, MARSHALL DANN Art est ing"Officer Commissioner of Patents USCOMM'DC 6D376P59 u U.S, GOVERNMENT PRINTING UFFIEE l99 0-366-334. 4 

